PROJECT SUMMARY The Phase I of this program met or exceeded the success criteria, implementing a miRNA assay and directly demonstrating single cell analysis on the Fluidigm C1 using a surrogate mRNA assay of 2700 genes. Having separately shown that TempO-Seq assay of FFPE correlates to matched frozen, this Phase II will address an NHGRI area of special interest Topic F ?Single cell genomic analysis?, establishing and validating a whole miRNome TempO-Seq assay based on miRBase 21, then validating and commercializing single cell TempO-Seq Open App assays on the Fluidigm C1 that measure the human whole transcriptome, a surrogate whole transcriptome supplemented with additional clinically relevant genes and mutations, and the whole miRNome. These assays will be benchmarked against bulk cell TempO-Seq assay, C1/BioMark HD qPCR assay, C1 Clontech SMARTer NGS assays, and RNAseq assay of bulk cells. Since the Fluidigm C1 is not the only commercial instrument that can provide single cell samples for testing, we will also develop and validate a microfluidic instrument (the CellCensus?) to perform the TempO-Seq assay using single cells separated by FACS or prepared on Silicon Biosystems DEPArray system. Using the DEPArray we will demonstrate the single cell assay of dissociated FFPE. The significance to investigators is that the TempO-Seq assays can provide the same coverage and as good or better sensitivity to measure low expressed genes as RNAseq but 10 to 100 more single cell samples can be sequenced for the same cost, which is important because more samples can be run to provide higher quality data accurate and sensitive molecular profiles to reliably identify subtypes of single cells despite the confounding impact of stochastic gene expression at the single cell level, and advance single cell research faster. The ability to perform single cell analysis on FFPE, exploiting the unique advantages of the DEPArray and TempO-Seq, will advance translational medicine by permitting the assay of single cells and identification of subpopulations from archived FFPE samples for which patient 5-, 10-, 20-year outcomes are known.